Increasing evidence suggests that cancer/tumor development is due to a rare population of cells, termed cancer stem cells (CSCs) (Dick, 2009; Jordan, 2009; Reya et al., 2001) that are uniquely able to initiate and sustain disease. In addition, experimental evidence indicates that conventional chemotherapeutics, characterized by their ability to inhibit cell proliferation of cancer cell lines (Shoemaker, 2006) or reduce tumor burden in murine models (Frese and Tuveson, 2007), are ineffective against human CSCs (Guan et al., 2003; Li et al., 2008). This resistance to chemotherapeutics is coupled with indiscriminate cytotoxicity that often affects healthy stem and progenitor cells, leading to dose restriction and necessitating supportive treatment (Smith et al., 2006). Recent examples along these lines include selective induction of apoptosis (Gupta et al., 2009; Raj et al., 2011) that remains to be tested in normal SCs and in the human system. Accordingly, the identification of agents that target CSCs alone is now critical to provide truly selective anti-cancer drugs for pre-clinical testing.
Normal and neoplastic SCs are functionally defined by a tightly controlled equilibrium between self-renewal vs. differentiation potential. In the case of CSCs, this equilibrium shifts towards enhanced self-renewal and survival leading to limited differentiation capacity that eventually allows for tumor growth. In contrast to direct toxic effects that equally affect normal SCs, an alternative approach to eradicate CSCs is by modification of this equilibrium in favor of differentiation in an effort to exhaust the CSC population. The identification of molecules that selectively target somatic CSCs while sparing healthy SC capacity would therefore be useful for the development of novel diagnostics and therapeutic treatments to selectively target human CSCs.
Methods of discovering anti-cancer compounds which have a selective effect on cancer cells compared to the compounds' effect on normal cells has been described (U.S. Pat. No. 6,180,357) in which anti-CSC drugs have been discovered. However the identification of CSCs require advances in technology. There are several advances required for discovering selective anti-CSC compounds. Some of these include growing high enough numbers of both CSC and normal stem cells (NSCs), having these cells stay in the non-differentiated state, having stem cells which are sufficiently robust for high throughput screening which include robotic handling, dispensing of solutions, transport, and development of relevant stem cell endpoints suitable for high throughput screening. The number of cells available for high throughput is a major technical limitation. Primary isolation is challenging since stem cells are rare in vivo and therefore culturing stem cells permits amplification of the number of stem cells available. Stem cells in culture tend to differentiate and not retain their stem cell characteristics in culture. An example of the difference between stem cell culture and general cell culture is that stem cell culture requires antibiotic free conditions to prevent differentiation. The cell number limitation is apparent in Kondo (WO2006051405A2) since the selection of side populations (as a surrogate of a CSC) by Hoechst 3334 is limiting, given that this dye is toxic to stem cells (Machaliński et al., 1998). In bulk culture, as disclosed in Kondo, a side population forms two populations of cells, a side population and a non-side population of cells. The culture does not maintain a pure group of side population cells and requires re-isolation of the side population with Hoechst 3334 thereby diminishing the number of cells. This issue also hinders determination of endpoints of the mixed cells in culture, since a pure population cannot be monitored directly. Further, subculture, and sub-population analysis is not possible because cell culture creates mixed populations. Although Kondo claims methods for discovering selective CSC compounds the inability to maintain pure cell populations, demonstrate normal cell effects by the same compounds, and compare them to the disclosed side population as a model of CSC in a high through put manner is a limitation of the current state of the art for discovering CSC selective drugs.
Likewise, Tyers (U.S. Pat. No. 8,058,243), illustrates other limitations in the current art. Tyers discloses a clonogenic neurosphere assay to identify potent and/or selective modulators of proliferation, differentiation and/or renewal of neural precursor cells, neural progenitor cells and/or self-renewing and multipotent neural stem cells. The screen was directed to compounds active in a stem cell assay, and not necessarily targeting a CSC. The counter screen was an astrocyte cell line rather than a normal stem cell line so that the counter screen is detecting stem cell vs. differentiated cell activity rather than selective CSC vs. NSC activity. From the active compounds they disclosed testing a subset of twelve (12) against medulloblastoma precursor cells which are enriched for CSCs but are not a pure population of CSCs. The discovery of anti-CSC compounds relied on finding active compounds against neurosphere cells and further testing. Since neurospheres contain stem cells and progenitor cells of the neural lineage the facility to discover anti-CSC selective compounds is precluded because the first step of their screen is directed to normal stem cells. Thus in Tyers the challenge of discovering anti-CSC compounds through high throughput is apparent since they teach identifying active stem cell compounds first through high throughput screening, then testing a sub-set on CSC enriched cells.
Hematological malignancies are types of cancer that affect blood, bone marrow and lymph nodes. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. Examples of myeloid malignancies include acute myeloid leukemia and chronic myeloid leukemia.
While myeloid malignancies are all generally considered to arise from precursors of the myeloid lineage in the bone marrow, they are highly divergent in presentation, pathology and treatment. For example, the 2008 World Health Organization Classification for Myeloproliferative Neoplasms (See Tefferi et al. Cancer, September 1st, pp. 3842-3847 (2009); also Vannucchi et al. Advances in Understanding and Management of Myeloproliferative Neoplasms CA Cancer J. Clin. 2009; 59:171-191, both hereby incorporated by reference), identifies 5 different classification schemes for myeloid neoplasms, and places acute myeloid leukemia (AML) in a separate category from chronic myelogenous leukemia (CML) and other myeloproliferative neoplasms. Furthermore, CML is often characterized as containing the BCR-Abl translocation which is absent in AML. Preferred treatments for leukemias, such as myeloid malignancies, would target leukemic cells without unduly affecting hematopoietic stem cell populations.
Thioridazine is a dopamine receptor antagonist that belongs to the phenothiazine drug group and is used as an anti-psychotic. It has been in clinical use since 1959, however because of concerns about cardiotoxicity and retinopathy at high doses this drug is not commonly prescribed, and is reserved for patients who have failed to respond to, or have contraindications for more widely used antipsychotics. Schizophrenic patients receiving dopamine receptor antagonist medication at doses deemed effective for schizophrenia have been reported to have a reduced incidence of rectum, colon, and prostate cancer compared to the general population.
There is a need for novel methods for the treatment and prognosis of cancers and in particular for novel methods for the treatment and prognosis of acute myeloid leukemia. There is also a need for novel methods for the identification and validation of agents that target cancer stem cells.